NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway
- Yichen Li†1,
- Bei Li†2,
- Xianzi Wan1,
- Wei Zhang1,
- Ling Zhong1Email author and
- Shao-Jun Tang2Email author
© Li et al; licensee BioMed Central Ltd. 2012
Received: 6 October 2011
Accepted: 4 January 2012
Published: 4 January 2012
Wnt proteins are emerging key regulators of the plasticity and functions of adult brains. However, the mechanisms by which the expression of Wnt proteins is regulated in neurons are unclear. Using cortical primary cultures, we show here that activation of NMDA receptors (NMDARs) induces rapid Wnt5a protein synthesis and secretion. This NMDAR-regulated Wnt5a synthesis does not require transcription and is a result of activity-dependent translation. We also show that NMDAR-regulated Wnt5a translation depends on MAPK signaling but not mTOR signaling. Our findings suggest that the synaptic activity of CNS neurons activates NMDARs, which in turn stimulate translation from stored Wnt5a mRNA via the MAPK signaling pathway.
Keywordsactivity-regulated protein synthesis Wnt protein mTOR signaling MAPK signaling NMDA receptors
Wnts are secreted glycoproteins that regulate cell morphologies and behaviors by stimulating complicate intracellular signaling cascades. Previous work has established that Wnt signaling controls many oncogenic and developmental processes [1, 2]. More recent studies have revealed that Wnt signaling is critically involved in key processes of the formation and plasticity of the nervous system, including neurogenesis , axon guidance , dendritic development , synaptic differentiation  and plasticity [7, 8]. Abnormalities of Wnt signaling are implicated in major brain disorders such as Alzheimer's disease [9–11], Parkinson's disease [12, 13], schizophrenia [14, 15], and drug abuse . Wnt5a is member of the Wnt protein family and plays important roles in outgrowth, guidance and branching of axons [17, 18]; genesis of dopaminergic neurons ; and formation and plasticity of both excitatory and inhibitory synapses [20–22]. Wnt5a administration was reported to improve specific pathological processes of Alzheimer's  and Parkinson's diseases in animal models .
Wnt proteins bind to receptors to activate the Wnt/β-catenin canonical pathway and β-catenin-independent non-canonical pathways, which include the planar cell polarity (PCP) pathway and the Wnt/calcium (Ca2+) pathway [2, 23–26]. In the canonical pathway, Wnts (such as Wnt3a) inhibit glycogen synthase kinase 3β (GSK-3β) and consequently stabilize β-catenin to regulate transcription . Wnt5a is a prototypic Wnt ligand that activates the non-canonical pathways [27, 28]. The activation of the PCP pathway stimulates Rho GTPases and c-Jun N-terminal kinase (JNK) to regulate cell morphogenesis and movement , whereas the activation of the Wnt/Ca2+ pathway causes Ca2+ to activate protein kinase C (PKC) and calcium/calmodulin dependent protein kinase II (CaMKII) . In neurons, Wnt secretion is intimately governed by synaptic activity, especially the activation of NMDA receptors (NMDAR) .
In contrast to the detailed understanding of the intracellular signaling cascades initiated by Wnts, little is known about the upstream mechanisms that control the synthesis of Wnt proteins. Wayman et al. recently showed that NMDAR activation stimulates CREB-mediated Wnt2 transcription .
We report here a mechanism that couples NMDAR activation to Wnt5a protein synthesis in primary cortical cultures. We observed that NMDAR activation elicited rapid increase and secretion of Wnt5a protein. This NMDAR-regulated Wnt5a protein increase was blocked by translational but not transcriptional inhibitors. In addition, mitogen-activated protein kinase (MAPK) but not mammalian target of rapamycin (mTOR) inhibitors abolished this Wnt5a synthesis. Our findings suggest that a NMDAR/MAPK pathway controls the activity-regulated translation of Wnt5a mRNA in cortical neurons.
NMDA receptor (NMDAR) activation rapidly increases Wnt5a in cortical cultures
NMDAR-elicited Wnt5a increase requires translation but not transcription
mTOR signaling pathway is not required for the NMDAR-dependent Wnt5a protein synthesis
NMDAR activation stimulates Wnt5a protein synthesis via the MAPK signaling pathway
Conclusion and Discussion
In this study, we found that NMDAR activation rapidly increases the synthesis of Wnt5a protein. We further elucidate that the NMDAR-regulated rapid Wnt5a synthesis depends on translation but not transcription and that NMDAR-induced translation from the preexisting Wnt5a mRNA is activated by MAPK signaling but not the mTOR signaling pathway.
Inestrosa and co-workers showed that Wnt5a modulates the plasticity of both glutamatergic and GABAergic synapses on hippocampal neurons [20, 21]. However, the mechanism of Wnt5a regulation during the induction and expression of synaptic plasticity was not known. Our findings reveal that synaptic activity, via NMDAR activation, stimulates the synthesis of Wnt5a protein. Because Wnt5a is in dendritic regions-near the presynaptic terminals in mature neurons (Figure 1A)-the rapid synthesis and secretion of Wnt5a following NMDAR activation probably provide an endogenous source of Wnt5a to alter the molecular organization and function of synapses. Indeed, Chen et al. reported that NMDAR-dependent secretion of Wnt3a regulates synaptic plasticity in hippocampal slices . These findings collectively support the view that activity-regulated synthesis and secretion of Wnts are basic molecular processes underlying the expression of synaptic plasticity .
The increase in NMDAR-regulated Wnt5a protein is a result of de novo translation that does not require mRNA transcription (Figure 2A, C). These findings indicate that there is dormant Wnt5a mRNA stored in neurons, and this mRNA is positioned for translational initiation following NMDAR activation. This provides a mechanism for neurons to quickly generate new Wnt5a, which is probably needed for synaptic processes that are critical in the early stage of synaptic plasticity soon after synaptic activation, including the re-organization of synaptic proteins [20, 21]. On the other hand, Wayman et al. showed that in differentiating hippocampal neurons NMDAR activation stimulates Wnt2 transcription, which regulates dendritic arborization . Together, these findings indicate that NMDARs may evoke the expression of different Wnt proteins by stimulating either transcription or translation in different cellular contexts.
The mTOR signaling pathway is a key mechanism by which synaptic activity stimulates protein synthesis in neurons [38, 39]. However, our results indicate that this pathway is not involved in the activation of NMDAR-regulated Wnt5a mRNA translation (Figure 4A, B). Instead, the NMDAR-elicited Wnt5a protein synthesis requires the activation of the MAPK signaling pathway. Tsokas et al. reported that MAPK signaling can stimulate activity-regulated synthesis of translational proteins by controlling the mTOR signaling pathway . Because mTOR is not required for Wnt5a synthesis (Figure 3), we conclude that MAPK signaling leads to translational activation via an mTOR signaling-independent pathway.
Materials and methods
NMDA [N-methyl-D-aspartic acid, product number (M3262)], DAP5 [D(-)-2-Amino-5-phosphonovaleric acid, A8054], Poly-D-lysine (P7280), U0126 (U120), Trypsin 10× solution (T4674), MSG (L-glutamic acid monosodium salt hydrate, G5889), Rapamycin (R8781), PD98059 (P215), Actinomycin D (A4262); Anisomycin (A9789) were purchased from Sigma; DAPI (s36939) from invitrogen; HBSS (Hank's balanced salt solution 10×, 14185), D-MEM/F-12 (Dulbecco's modified eagle medium: nutrient mixture F-12, 12400-024), L-Glutamine 100× (25030), B27 50×, NBM (Neurobasal medium, 21103) from Gibco; FBS (Fetal bovine serum, A15-101) from PAA; and DMSO (0231) from Amresco.
NMDA was dissolved in NBM 5 min before treatment. DAP5, U0126, Rapamycin, PD98059, Anisomycin were prepared as 1000× concentrated stocks in DMSO. All other compounds were prepared as 1000× concentrated stocks in ultrapure water.
Anti-Wnt5a antibody was purchased from R&D Systems (AF654); anti-p-P70S6K (Thr389) antibody from Cell Signaling Technology (#9206); anti-GAPDH antibody from Santa Cruz (SC-32233); anti-Synapsin I from Millipore (AB1543P); and FITC-conjugated donkey anti-rabbit secondary antibody (711-097-020) and Rhodamine-conjugated rabbit anti-goat secondary antibody (305-297-003) from Jackson.
Primary cortical culture
Cortical cultures were prepared as described (32). Briefly, cortices were dissected from C57BL/6J mouse embryos (E18) in HBSS, stripped from blood vessels, and cut into small pieces. They were then digested in 1× trypsin for 8 min at 37° in 5 ml tubes and dissociated into single cells by gentle aspirations with a fire-polished glass pipette. After sitting on the bench for 2 min, cells in the supernatant were transferred into fresh tubes and centrifuged for 5 min (1000 rpm). Cell pellets were suspended in DMEM. Cells were plated on 12-well plates (JETBIOFIL) with poly-D-lysine (20 μg/ml) at a density of 5 × 105 cells/well and incubated at 37° in a humidified atmosphere of 95% air and 5% CO2. One hour later, the culture media were replaced with NBM supplemented with 2% B27, 5 mM glutamine, 1% streptomycin and penicillin. The media were changed every three days. Cultures were used for stimulation at day 10 in vitro.
Real-time fluorescence quantitative PCR
Cultures (10 DIV; 2 × 106 cells/well in 6-well plates) were switched to fresh media for 1 h and then stimulated with NMDA (50 μM) for 15 min at 37°. Total RNA was purified from the cultures with TRIZOL (invitrogen) according to the manufacturer's instructions. The RNA purity was determined by the OD260/OD280 ratio, and the concentration was calculated based on OD260. The RNA (1.5 μg) was used for reverse transcription, followed by quantitative real-time PCR using PrimeScriptTM RT reagent kit (TaKaRa). PCRs (25 μl reactions) contained 12.5 μl 2× SYBR Premix Ex TaqTM, 0.5 μl PCR Forward Primer (10 μM), 0.5 μl PCR Reverse Primer (10 μM), 9.5 μl dH2O and 2 μl cDNA. The following primers were employed: Wnt5a Reverse primer: 5'-AGCCAGCACGTCTTGAGGCTA-3'; Wnt5a Forward primer: 5'-AA TCCACGCTAAGGGTTCCTATGAG-3'; β-actin Reverse: 5'-GCAATGCCTGGGTACATGGTGG-3'; and β-actin Forward: 5'-ACGCGTCGACCTCCTTGCAGTCCATTTT-3'. PCR was run for one cycle at 95° for 10 s, and 40 cycles at 95° for 5 s; 60° for 20 s.
Primary cortical neurons that had been grown on glass coverslips were briefly washed twice with cold PBS, and then fixed in 4% paraformaldehyde for 30 min at room temperature (RT). Neurons after fixation were washed with cold PBS (3 × 5 min), permeabilized with 0.1% Triton X-100 for 10 min, rinsed three times, and blocked with 1% BSA in PBS for 1 h (RT). Next, neurons were incubated with primary antibodies (double-stained with anti-Wnt5a antibody at 1:50 and anti-synapsin I antibody at 1:200) in 1% BSA/PBS in a humidified chamber overnight at 4°, rinsed three times in PBS (3 × 5 min). This was followed by incubation with secondary antibodies [Rhodamine-conjugated anti rabbit IgG (recognizing anti-Wnt5a antibody) and FITC-conjugated anti goat IgG (recognizing anti-synapsin I antibody)] in 1% BSA/PBS in a light-proof container (1 h at RT). Then, cells were washed (3 × 5 min in PBS), stained with 0.1 μg/ml Hoechst for 1 min, and rinsed with PBS before being mounted.
To detect intracellular proteins, cortical neurons in 12-well plates (5 × 105 cells/well; 10 DIV) were rinsed with PBS and lysed immediately in 100 μl of 2× SDS-PAGE sample buffer (1×: 62.5 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue as described previously ). These were then boiled for 10 min. After electrophoresis on 10% SDS-PAGE gels, proteins were transferred to 0.2 μm Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore) and blotted with primary and HRP-conjugated secondary antibodies. The signals were detected using the ECL system (Pierce). To detect secreted Wnt5a, media of cortical neurons in 12-well plates (1 × 106 cells/well; 10 DIV) were replaced with 300 μl NBM before NMDA stimulation. All NBM was collected after the stimulation and heat-evaporated to a final volume suitable for one loading on an SDS-PAGE gel.
Quantification and statistics
Immunoblots were scanned with an Epson scanner, and the optical density (OD) of protein bands were quantified with Quantity One software (Bio-Rad). The statistical tests were performed by one-way ANOVA or by two-tailed Student's tests, using SPSS 16.0. Graphs of quantified data (Mean data + SEM) were prepared using Origin.
This work was supported by the UTMB start-up funds and the Whitehall Foundation to S.-J. T., the National Natural Science Foundation of China (No. 30873457 to L. Z. and No. 30873457 to W. Z.) and the Scientific Technology Project of Guangdong Province of China (No.2008A060202010 and No. 2010B050700019 to L. Z. and 2008A060202010 and 2010B050700019 to W.Z.).
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